This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Monoclonal antibodies (mAbs) as therapeutic agents are recognized for their specificity, and their ability to elicit an immune response, antagonize signaling pathways, or as vehicles to deliver cytotoxic compounds at disease site. While these targeted approaches show improved PK/PD properties, many mAbs recognize antigens that are of human origin. At therapeutic doses, these mAbs bind to antigen expressed on normal tissue and can produce adverse side effects. To address these adverse side effects, we have recently developed a method to modulate the antigen affinity of therapeutic mAbs and use a tumor-associated protease to activate the mAb at the disease site. We show that cleavage of the 'pro-antibody'restores the mAb antigen affinity. Herein, we are seeking to identify novel interactions within the Fab framework region of the mAbs using diffraction methods. Successful identification of these molecules will permit rapid optimization of our current design, facilitate clinical trials and may significantly improve this line of therapy.